MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in plants and animals. In plants, they are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNAs. Although plant miRNAs have some conserved functions extending beyond development, the importance of miRNA-directed gene regulation during plant development is now becoming clear miRNAs are already known to play numerous crucial roles at each major stage of development, typically at the core of gene regulatory networks, targeting genes that are themselves regulators. So far, microRNAs have been found to be involved in plant development, regulation of abiotic and biotic stress responses and hormone signaling (Jones-Rhoades et al., 2006, Ann. Rev. Plant Biol., 57:19-53).
A commonly-used approach in identifying the function of novel genes is through loss-of-function mutant screening. In many cases, functional redundancy exists between genes that are members of the same family. When this happens, a mutation in one gene member might have a reduced or even non-existing phenotype and the mutant lines might not be identified in the screening.
Using miRNAs, multiple members of the same gene family can be silenced simultaneously, giving rise to much more intense phenotypes. This approach is also superior to RNA interference (RNAi) techniques, in which typically 100-800 bp fragments of the gene of interest form a fold-back structure when expressed. These long fold-back RNAs form many different small RNAs and prediction of small RNA targets other than the perfectly complementary intended targets is therefore very difficult. miRNAs, in contrast, are produced from precursors, which are normally processed such that preferentially one single, stable small RNA is generated, thus significantly minimizing the “off-target” effect.
A second approach to functional screening is through over-expression of genes of interest and testing for their phenotypes. In many cases, attempting to over-express a gene which is under miRNA regulation results in no significant increase in the gene transcript. This can be overcome either by expressing a miRNA-resistant version of the gene or by down-regulating the miRNA itself.
Taste characteristics are a major determinant of fruit quality for both processing and fresh market tomatoes (see Stevens, M. A., 1986, “Inheritance of tomato fruit quality components,” Plant Breeding Reviews, 4: 274-310). One of the major components of taste in tomatoes is soluble sugar content. The soluble sugar content of all known commercial cultivars of tomatoes primarily includes the hexose sugars glucose and fructose in near-equimolar ratios (1:1 to 1:1.3). In commercial tomato cultivars, the disaccharide sucrose is also present but at concentrations rarely exceeding 0.5% on a fresh weight basis. Certain wild species of tomato accumulate high concentrations of sucrose, which may reach 4% on a fresh weight basis. In the presence of high sucrose, these fruit accumulate low levels of the hexoses fructose and glucose, typically less than 1% each on a fresh weight basis and the ratio of fructose to glucose is unusually high, more than 1.5:1.
Typically, plant breeders seek to improve the sweetness component of tomato flavor by increasing total soluble solids (TSS) measured by refractometric determination of a sample of juice and expressed as Brix. This measurement, however, does not differentiate between the component sugars. Fructose is significantly sweeter than both glucose and sucrose giving a tomato with a relatively high fructose content distinct advantage in terms of superior taste characteristics.